Cytochrome P450 pharmacogenetics : implications for anticancer and warfarin therapy

Abstract: There is a pronounced interindividual variability in the drug disposition, response and toxicity. Pharmacogenetics aims at identifying genetic biomarkers that could help to increase the drug efficacy, reduce adverse drug reactions and contribute to the development of personalized medicine. Polymorphic cytochrome P450 genes encoding heme-containing ER membrane bound monooxygenases that metabolize xenobiotics, drugs and also endogenous compounds, strongly contribute to interindividual variations in drug response. In the present work we have investigated molecular mechanisms of the adverse drug reactions caused by the polymorphic changes in the cytochrome P450 2C8 (CYP2C8), CYP2C9 and CYP3A4 genes and, in addition developed a novel enzymatic assay for CYP2W1. CYP2W1, a P450 enzyme mainly expressed in colon cancer, has an unknown function and no specific substrates were previously identified. Despite the unusual inverse membrane topology of CYP2W1 that allows its glycosylation but prevents interaction with the redox partner, P450 oxidoreductase (POR), we discovered specific CYP2W1-mediated metabolism of indolines, which indicates the presence of a yet unknown electron transport chain in the lumen of ER. CYP2C9 catalyzes the metabolism of anticoagulant drug warfarin. We characterized the newly discovered rare CYP2C9'35 allele encoding an enzyme with two amino acid changes including Arg125Leu that was found in a patient with warfarin hypersensitivity. The expression of the variant proteins in the mammalian HEK293 cell system showed abolished activity of the CYP2C9.35 enzyme towards warfarin in NADPH supported reaction, but the enzyme could be activated when NADPH was replaced by hydroperoxides. This indicates that CYP2C9.35 is unable to receive electrons from POR because of the impaired interaction with this redox partner. Indeed, in silico modeling confirmed this conclusion showing disrupted salt bridges between CYP2C9.35 and POR due to the mutation of key residues involved in such interaction. CYP3A4 is one of the key enzymes, which metabolizes the anticancer drug paclitaxel. In a cohort of 236 Spanish patients with a paclitaxel induced neuropathy whole exome sequencing revealed the presence of different rare CYP3A4 gene variants, CYP3A4'8, CYP3A4'20, CYP3A4'25 (p.Pro389Ser) and CYP3A4'27 (p.Leu475Val), the latter two previously not described. The expression of these two novel gene variants in HEK293 cells revealed that the corresponding enzymes are more unstable than the CYP3A4.1 enzyme and carriers of these rare CYP3A4 variants had much higher risk for neuropathy and a need in paclitaxel treatment modifications. The data indicate enrichment of these rare defect CYP3A4 alleles in the group of the paclitaxel induced neuropathy patients and suggest that genotyping of CYP3A4 defective variants may provide a basis for paclitaxel treatment individualization. Based on previous data indicating a role for the defective CYP2C8'3 allele for paclitaxel induced neuropathy, we also investigated the influence of this polymorphism on paclitaxel induced neuropathy and neuropathy risk in 148 patients receiving paclitaxel as well as the CYP2C8.3 catalyzed metabolism of paclitaxel in a mammalian expression system. However, in contrast to many other studies we found no significant effect of this allele on paclitaxel induced neuropathy or paclitaxel metabolism in vitro. In conclusion, our data indicate the importance of rare genetic CYP variants for induction of selective drug induced adverse reactions and emphasize the necessity of more extensive genetic analyses, e.g. whole exome sequencing, before fully individualized drug therapy can be achieved.